An outstanding goal in evolutionary biology is to understand the mechanisms underlying phenotypic diversity, both within and between species. This involves identifying both the processes that drivechange (including forcessuch as mutation, selection, and drift) and the substrates that those processes act upon (at molecular, genetic, and organismal levels). Developing a full picture of the evolutionary process requires a system that is both variable and tractable. Pigmentation is an effective model for integrating phenotype, genotype, and molecular function in an evolutionaryframework. The virilis group of Drosophila is an emerging focusof evolutionary study. Drosophila novamexicanaand D. americana are sister taxa within this group (0.38 MYdivergence) that currently have nonoveriapping ranges in the United States. They have recently evolved light abdominal pigmentation, which is fixed in D. novamexicana and forms a dine in D. americana. Flies on the western side of the D. americana range are lightly colored, while flies on the eastern side are darkly colored. The dine exists in the absence ofany detectable population structure at neutrally evolving lod, suggesting maintenance by natural selection. Fine scale genetic mapping has identified the first intron of the tan gene as contributing to intra- and interspecific pigment variation. In Specific Aim 1of this project, a transgenic approachwill be usedto determine which specific nudeotide substitutions in the tan gene are causal. This will constitute one of few cases in whichthe molecular basisof a major evolutionary change isknown. Any single molecular analysis may capture only a small fraction of the existing variation. Aim 2 takesa genomic approach to obtaining a complete picture of sequence variation at tan relative to the rest of the D. americana genome. Association mapping will be usedto test the universality of the putatively causalvariant in Intron 1of tan. Fst and dinal analysis will be used to identify any evidence of selection on tan. Aim 2 generates a rare view into the quantity and geographic distribution of standing variation at a phenotypically relevant locus, as well as the role of selection in maintaining the observedgenetic patterns. A major breakthrough in human genomics is the revelation of the vastness of individual genetic variation, which poses a challenge to modem medidne because manyvariants have important effectson patients'disease susceptibility and responseto treatment. D. americana has a large effective population size and high genetic diversity, making it ideal for studying the genetics of individual differences. The proposedstudy will reveal both the molecular basis of variation and the dynamics of these variants in natural populations.